Fig 2: Mutation of K772 and K798 abolishes the rescue of a mitotic delay after knockdown of APC4. a HeLa cells stably expressing GFP-tagged Histone 2B and containing inducible constructs of either APC4 wild type or K772,798R mutant were infected with lentivirus expressing either a control shRNA or an shRNA raised against endogenous APC4. Knockdown of the inducible APC4 constructs was abolished by introducing point mutations at the sites of shRNA binding. Two days after infection with the viruses and induction of the APC4 constructs, the time needed for mitotic progression from metaphase to anaphase onset was monitored by live cell imaging. The mean value and the SEMs are shown for 70 cells per condition and p-values were obtained via Student’s t-test. p-values: *p < 0.02, **p < 0.005. b Knockdown of APC4 was confirmed via western blot analysis of cell lysates 2 days after infection with the lentiviruses expressing either control shRNA or shRNA against APC4. The blot was stained with Ponceau S to monitor equal sample loading

Fig 3: APC4KR-SUMO rescues APC4KR metaphase-anaphase delay.(A) A construct coding for a FLAG-tagged APC4KR-SUMO2 fusion protein (APC4KR-S2) was used to generate stable inducible lines in YFP-H2B HeLa cells. (B) Cells were transfected with control or APC4-targeting siRNAs and cultured in the presence (+Dox) or absence of doxycycline. Endogenous APC4 and FLAG-APC4KR-S2 expression levels were analyzed by immunoblot analysis with APC4 and tubulin specific antibodies. (C) Representative cells from timelapse acquisition beginning with NEBD to anaphase onset in control and FLAG-APC4KR-S2 expressing cells with timestamps indicated in minutes. (D) Mitotic timing beginning with NEBD to metaphase plate alignment and from metaphase plate alignment to anaphase onset was quantified in control and FLAG-APC4KR-S2-expressing cells. Experiments were performed in triplicate; means are displayed and error bars represent standard deviations. n = 60 for each cell line. Two-tailed t-tests were used to calculate significance: p=0.16 between control and FLAG-APC4KR-S2 timing from NEBD-metaphase, p=0.35 for control and FLAG-APC4KR-S2 timing from metaphase plate alignment to anaphase. (E) Individual metaphase to anaphase transition times are displayed for control and FLAG-APC4KR-S2-expressing cells. n = 314 for each cell line.

Fig 4: pUL21a reduces APC4 and APC5 protein levels and inhibits APC activity in an inducible cell line.(A) pUL21a expression is sufficient to reduce APC4 and 5 protein accumulation. Hela cells constitutively expressing GFP-tagged TetR were transduced with pLKO-derived lentivirus that expressed pUL21aPH-AA, pUL21astop, or pUL21aPR-AA under a Tet operator (TetO)-regulated CMV promoter. Transduced cells were enriched by antibiotic selection and pUL21a expression was induced by tetracycline treatment for 72 hours. Cell lysates were collected and analyzed by immunoblotting. (B) pUL21a expression is sufficient to induce an M-phase cell cycle arrest. Cells carrying pUL21aPH-AA (+/− tetracycline) were treated with nocodazole (100 ng/µl) for 16 hours to synchronize cells in G2/M phase. The cells were then released from nocodazole, collected at indicated time points, and analyzed for DNA content by flow cytometry. (C) pUL21a expression is sufficient to regulate APC activity. Cells from panel B were also analyzed for APC subunit and substrate levels by immunoblotting.

Fig 5: Working model of virus-mediated APC regulation during HCMV infection.HCMV uses two mechanisms to regulate the APC during infection. The pUL97 viral kinase inhibits the APC co-activator Cdh1 by phosphorylation while pUL21a targets APC4 and APC5 for proteasome-dependent degradation to dissociate the complex.